Hydrolase-catalyzed biotransformations in deep eutectic solvents

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Johnathan T. Gorkeab, Friedrich Srienc*ab and Romas J. Kazlauskas*bc
aDepartment of Chemical Engineering and Materials Science, University of Minnesota, 151 Amundson Hall, 421 Washington Ave. SE, Minneapolis, MN 55455, USA. E-mail: srienc@umn.edu; Fax: +1 612 626 7246; Tel: +1 612 625 6362
bBioTechnology Institute, 240 Gortner Lab, University of Minnesota, 1479 Gortner Ave., Saint Paul, MN 55108, USA. Fax: +1 612 625 1700; Tel: +1 612 624 9776
cDepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 140 Gortner Lab, 1479 Gortner Ave., Saint Paul, MN 55108, USA. E-mail: rjk@umn.edu; Fax: +1 612 625 5780; Tel: +1 612 624 5904

Received (in Maryland, MD) 7th November 2007, Accepted 14th January 2008

First published on the web 31st January 2008

Hydrolases show good catalytic activity in deep eutectic solvents, despite the presence of urea, which can denature enzymes, or alcohols, which can interfere with hydrolase-catalyzed reactions.

Room-temperature ionic liquids are potential green alternatives to organic solvents1 for extractions,2 chemical reactions3 and biotransformations.4 Ionic liquids are non-volatile, thermally stable and their solvation properties vary by changing the cation and anion. The limitations of ionic liquids are cost (240 $US kg−1 for 95% pure 1-butyl-3-methylimidazolium chloride vs. 30 $US kg−1 for 99% pure acetone or toluene), toxicity similar to or higher than organic solvents,5 and the need for high purity, as even trace impurities affect physical properties.6

Deep eutectic solvents (DESs)—eutectic mixtures of an ammonium salt and a hydrogen-bond donor such as choline chloride and urea—are alternatives to ionic liquids.7 Like ionic liquids, DESs often have melting points below room temperature, low volatility, and high thermal stability; but unlike ionic liquids, they include uncharged components—urea in the example above. No one has yet reported biotransformations in DESs, likely because strong hydrogen-bond donors like urea denature proteins. For example, 10 M urea or 5 M choline chloride inactivate lipase B from Candida antarctica (70% or 25% loss in activity after 90 min at 60 °C, respectively). DESs also contain halides, which inactivate or inhibit proteins when present in ionic liquids.8 In spite of these possible problems, we found that many hydrolases retain excellent activity in DESs.

As a test reaction, we used the lipase-catalyzed transesterification of ethyl valerate with 1-butanol, Table 1. CALB§ and the immobilized form—iCALB—catalyzed transesterification in all eight DESs tested and showed conversions comparable to that in toluene for five of the DESs. CALA also showed activity in all eight DESs, but showed conversions comparable to toluene only in ChCl:Gly (76 vs. 70%). PCL showed lower conversions than the other enzymes, but the conversion in one DES—ChCl:Gly—was higher than that in toluene (22 vs. 5%). The transesterification activity of CALB did not decrease in ChCl:U at 60 °C over 90 min, making it at least 20- to 35-fold more stable in the mixture than in aqueous solution of the components: 10 M urea or 5 M choline chloride (see ESI).


Table 1 Percentage conversion of ethyl valerate to butyl valerate at 60 °C
iCALB
 CALB
 CALA
 PCL
 No enzyme
ChCl:Acet 23a 96 0.5 0.0 0.0
ChCl:EG 11 (99)b 32 (93)b 3.0 0.2 0.0
ChCl:Gly 96 96 70 22 0.0
ChCl:MA 30 58 0.7 0.0 0.7
ChCl:U 93 99 1.6 0.8 0.0
EAC:Acet 63 92 2.7 0.0 0.0
EAC:EG 23 (54)b 33 (79)b 20 0.0 0.0
EAC:Gly 93 91 2.1 0.5 0.0
Toluene 92 92 76 5.0 0.0
a 40 mM ethyl valerate, 400 mM 1-butanol, 10 mg ml−1 enzyme, 24 h.  b Number in parentheses is the percentage conversion including the side reaction with the ethylene glycol component of the deep eutectic solvent. No side reaction was detected in the other reactions.  

Some DESs contain an alcohol component—ethylene glycol or glycerol, which competes with 1-butanol in transesterification. Indeed, ethylene glycol monoester was the major product for reaction in the two DESs containing ethylene glycol. For example, the CALB-catalyzed reaction in EAC:EG showed 54% of starting material consumed resulting in 31% ethylene glycol monovalerate and 23% ethyl butyrate. This nearly equal amount of the two product esters is surprising because the concentration of ethylene glycol (10 M) was 25 times higher than the concentration of 1-butanol (400 mM). In a competition between ethylene glycol and 1-butanol in tert-butanol, 1-butanol reacted three times faster. Thus, ethylene glycol was 9-fold less reactive in transesterification when it was present as a component of a DES.

Even more surprising were the transesterification reactions in glycerol-containing DESs (8 M glycerol) which showed >90% conversion and <0.5% glyceryl ester formation (see ESI). In a competition between glycerol and 1-butanol in tert-butanol, 1-butanol reacted six times faster. Thus, glycerol was >600-fold less reactive in transesterification when it was present as a component of a DES.

The initial specific activity for iCALB-catalyzed transesterification was comparable or higher in DESs as compared to typical ionic liquids, Table 2. Previous work showed that iCALB had good transesterification activity in 1-butyl-3-methylimidazolium bis(trifluoromethane)sulfonimide (BMIM [Tf2N]) compared to other ionic liquids.9 1-Butyl-3-methylimidazolium tetrafluoroborate (BMIM[BF4]) is water miscible like the DESs and preserves enzyme activity.10 iCALB had the highest initial specific transesterification activity in EAC:Gly (50 µmol ethyl valerate h−1 mg−1), which was twice as high as for BMIM[Tf2N] (24 µmol h−1 mg−1) and seven times higher than the activity we found in BMIM[BF4] (7 µmol h−1 mg−1). The initial specific activity in toluene, ChCl:Gly, and ChCl:U ranged from 20 to 37 µmol h−1 mg−1 and were comparable to that in BMIM[Tf2N].


Table 2 Initial specific activity of iCALB after 15 min of reaction
Transesterification activitya
Aminolysis activitya
Toluene 37 (100%) 46 (100%)
ChCl:Gly 33 (89%) 52 (113%)
ChCl:U 20 (54%) 22 (48%)
EAC:Gly 50 (135%) Not determinedb
BMIM[Tf2N] 24 (65%) 11 (24%)
BMIM[BF4] 7 (19%) 9 (19%)
a µmol ethyl valerate h−1 mg solid−1, activity relative to toluene in parentheses. iCALB contains 10 wt% protein.  b Aminolysis with ethylamine from the DES was the dominant reaction.  

iCALB also catalyzed another test reaction in DESs—aminolysis of ethyl valerate with 1-butylamine, Fig. 1. The reaction rates and final conversion (>90%) were similar in ChCl:Gly, ChCl:U or toluene. Aminolysis was slower in ChCl:Acet and gave only 39% conversion. In EAC:Gly aminolysis with ethylamine from the DES (4.5 M amine, 80% conversion) predominated over aminolysis with butylamine (11% conversion), likely due to proton exchange between butylamine with the ethylammonium cation. The relative amount of ethylamide formed is seven-fold less than the relative concentration of ethylamine. In contrast, the transesterification reactions above did not show competing aminolysis when using ethylammonium-chloride-containing DESs. A possible reason is that the ethylammonium ion remained protonated and unreactive because the transesterification reactions lacked a base.


Fig. 1 Aminolysis of ethyl valerate (100 mM) with 1-butylamine (110 mM) at 60 °C with 10 mg ml−1 iCALB

The initial specific activity for aminolysis in DESs was also higher than in ionic liquids, Table 2. The aminolysis activity in ChCl:Gly (52) was five times higher than in BMIM[BF4] or BMIM[Tf2N] (9–11) and similar to the activity in toluene (46 µmol h−1 mg−1).

DESs were also suitable as cosolvents for reactions in aqueous solution, where they enhanced hydrolase-catalyzed reactions up to 20-fold. The rates of esterase-catalyzed hydrolysis of p-nitrophenyl acetate increased moderately upon addition of 10 vol% ChCl:Gly: three-fold increase for PLE and ROE and a 25% increase for PFE and CALB (see ESI) The rate of epoxide hydrolase11 catalyzed hydrolysis of styrene oxide increased dramatically: a 20-fold increase in conversion, Table 3. The conversion was only 4.6% in buffer, but increased to 92% upon addition of 25 vol% ChCl:Gly, with no change in the enantioselectivity (E = 16; Janssens group has created a mutant with higher enantioselectivity, but we have not tested whether it shows a similar increase in conversion.12) Similar additions of 10 or 25% DMSO or acetonitrile did not increase the conversion for epoxide hydrolase-catalyzed hydrolysis of styrene oxide, suggesting that the effect is not a simple increase in substrate solubility. Adding more than 25 vol% DES decreased conversions. For example, the conversion of the EHAD1-catalyzed reaction decreased from 92% conversion in 25 vol% DES to 2% in 50 vol% DES. Similarly the rates of esterase-catalyzed hydrolysis of p-nitrophenyl acetate decreased in solutions containing more than 25 vol% DES (Fig. S2 in ESI).


Table 3 Percentage conversion of styrene oxide by EHAD1 in ChCl:Gly/buffer mixtures at 37 °C after 2 h
Cosolvent
 90%a
75%
 50%
 25%
 10%
 0%
ChCl:Gly 0 0 2.0 92 36 4.6
DMSOb N.D. N.D. N.D. 0.7 1.9 4.6
Acetonitrile N.D. N.D. N.D. 0.1 0.7 4.6
a Cosolvent volume faction in solution containing 0.05 mg ml−1 enzyme, 100 mM styrene oxide at 5 mM BES at pH 7.2 used as buffer.  b DMSO or acetonitrile added in place of DES.  

The polarity of these DESs is higher than typical imidazolium-based ionic liquids according to the Reichardts dye method, Table 4. The ETN of the DESs ranged from 0.77 to 0.93 as compared to 0.53 to 0.75 for imidazolium-based ionic liquids.13 The polarity of DESs containing the quaternary salt choline chloride was 0.08 to 0.09 units lower than the corresponding primary ethylammonium chloride DESs, which is consistent with the lower polarity of quaternary ammonium-based ionic liquids as compared to primary ammonium-based ionic liquids.13


Table 4 Solvent polarity according to color of dissolved Reichardts dye
Solvent
 ETN
Solvent
 ETN
Toluene 0.1014a ChCl:EG 0.80
DMSO 0.4414 Glycerol 0.8114
Acetonitrile 0.4614 ChCl:Gly 0.84
BMIM[Tf2N] 0.6413 ChCl:U 0.84
BMIM[BF4] 0.6813 EAC:Acet 0.85
Methanol 0.7614 EAC:EG 0.88
ChCl:Acet 0.77 EAC:Gly 0.93
a Solvent polarity according to Reichardts normalized polarity scale, where water has a polarity of 1.0 and trimethylsilane has a polarity of 0.0.  

The components within the DESs are 20- to >600-fold less reactive than expected based on their concentration in denaturation and in transesterification reactions. This lowered reactivity corresponds to 2–4 kcal mol−1, similar to the energy associated with the formation of hydrogen bonds. We propose that the hydrogen-bond network in DESs lowers the chemical potential of the components of DESs and thereby makes them suitable as solvents for a much wider range of reactions than one would predict based only on the components. The DES components are inexpensive (e.g., 65 $US kg−1 for choline chloride, 20 $US kg−1 for urea, 35 $US kg−1 for glycerol) and biodegradable, making DESs attractive as green replacements for volatile organic solvents.

We thank the National Institutes of Health Biotechnology Training Grant and the University of Minnesota Initiative for Renewable Energy and the Environment for funding.

Notes and references

1(a) T. Welton, Chem. Rev., 1999, 99, 2071 [Links]; (b) P. Wasserscheid and W. Keim, Angew. Chem., Int. Ed., 2000, 39, 3772 [Links]; (c) P. Wasserscheid and T. Welton, Ionic Liquids in Synthesis, Wiley-VCH, Weinheim, 2003.
2(a) J. G. Huddleston, A. E. Visser, R. P. Swatlowski, W. M. Reichert, S. T. Griffin and R. D. Rogers, Ind. Eng. Chem. Res., 2000, 29, 3596 [Links]; (b) H. Zhao, S. Xia and P. Ma, J. Chem. Technol. Biotechnol., 2005, 80, 1089 [Links].
3(a) H. Zhao and S. V. Moltrova, Aldrichimica Acta, 2002, 35, 75 [Links]; (b) C. M. Gordon, Appl. Catal., A, 2001, 222, 101 [Links]; (c) N. Jain, A. Kumar, S. Chauhan and S. M. S. Chauhan, Tetrahedron, 2005, 61, 1015 [Links].
4(a) R. M. Lau, F. van Rantwijk, K. R. Seddon and R. A. Sheldon, Org. Lett., 2000, 2, 4189 [Links]; (b) P. Lozano, T. De Diego, D. Carrié, M. Vaultier and J. L. Iborra, Biotechnol. Lett., 2001, 23, 1529 [Links]; (c) U. Kragl, M. Eckstein and N. Kaftzik, Curr. Opin. Biotechnol., 2002, 13, 565 [Links]; (d) R. A. Sheldon, R. M. Lau, M. J. Sorgedrager, F. van Rantwijk and K. R. Seddon, Green Chem., 2002, 4, 147 [Links]; (e) S. Park and R. J. Kazlauskas, Curr. Opin. Biotechnol., 2003, 14, 432 [Links].
5(a) B. Jastorff, R. Störmann, J. Ranke, K. Mölter, F. Stock, B. Oberheitmann, W. Hoffmann, J. Hoffmann, M. Nüchter, B. Ondruschka and J. Filser, Green Chem., 2003, 5, 136 [Links]; (b) J. Ranke, K. Mölter, F. Stock, U. Bottin-Weber, J. Poczobutt, J. Hoffmann, B. Ondruschka, J. Filser and B. Jastorff, Ecotoxicol. Environ. Safety, 2004, 58, 396 [Links]; (c) S. Stolte, J. Arning, U. Bottin-Weber, M. Matzke, F. Stock, K. Thiele, M. Uerdingen, U. Welz-Biermann, B. Jastorff and J. Ranke, Green Chem., 2006, 8, 621 [Links].
6(a) K. R. Seddon, A. Stark and M. Torres, Pure Appl. Chem., 2000, 72, 2275 [Links]; (b) A. K. Burrell, R. E. Del Sesto, S. N. Baker, T. M. McCleskey and G. A. Baker, Green Chem., 2007, 9, 449 [Links].
7(a) A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed and V. Tambyrajah, Chem. Commun., 2003, 70 [Links]; (b) A. P. Abbott, D. Boothby G. Capper, D. L. Davies and R. K. Rasheed, J. Am. Chem. Soc., 2004, 126, 9142 [Links]; (c) A. P. Abbott, G. Capper, D. L. Davies, K. J. McKenzie and S. U. Obi, J. Chem. Eng. Data, 2006, 51, 1280 [Links]; (d) A. P. Abbott, G. Capper, K. J. McKenzie and K. S. Ryder, J. Electroanal. Chem., 2007, 599, 288 [Links]; (e) A. P. Abbott, P. M. Cullis, M. J. Gibson, R. C. Harris and E. Raven, Green Chem., 2007, 9, 868 [Links].
8S. H. Lee, S. H. Ha, S. B. Lee and Y. M. Koo, Biotechnol. Lett., 2006, 28, 1335 [Links].
9J. T. Gorke, K. Okrasa, A. Louwagie, R. J. Kazlauskas and F. Srienc, J. Biotechnol., 2007, 137, 306 [Links].
10S. Park and R. J. Kazlauskas, J. Org. Chem., 2001, 66, 8395 [Links].
11(a) J. H. L. Spelberg, R. Rink, R. M. Kellogg and D. B. Janssen, Tetrahedron: Asymmetry, 1998, 9, 459 [Links]; (b) R. Rink and D. B. Janssen, Biochemistry, 1998, 37, 18119 [Links].
12(a) R. Lingun, L. Cao, W. Chen, K. F. Reardon and T. K. Wood, Appl. Environ. Microbiol., 2005, 71, 3995 [Links]; (b) R. Rink, J. H. L. Spelberg, D. B. Janssen and R. Furstoss, Tetrahedron, 2001, 57, 2775 [Links].
13C. Reichardt, Green Chem., 2005, 7, 339 [Links].
14C. Reichardt, Chem. Rev., 1994, 94, 2319 [Links].

Footnotes
Electronic supplementary information (ESI) available: Experimental procedures and supporting data. See DOI: 10.1039/b716317g
Synthesis of deep eutectic solvents: ammonium salt (0.05 mol) and hydrogen-bond donor (0.1 mol for choline chloride mixtures, 0.075 mol for ethylammonium chloride mixtures) were added to a 20-ml vial and heated at 80 °C until a clear, homogenous liquid formed, typically 1 h.
§ Abbreviations of deep eutectic solvents: ChCl:Acet = choline chloride–acetamide; ChCl:EG = choline chloride–ethylene glycol; ChCl:Gly = choline chloride–glycerol; ChCl:U = choline chloride–urea; ChCl:MA = choline chloride–malonic acid; EAC:Acet = ethylammonium chloride–acetamide; EAC:EG = ethylammonium chloride–ethylene glycol; EAC:Gly = ethylammonium chloride–glycerol.Abbreviations of enzymes: CALA = Roche. Chirazyme L-5 (lyophilized Candida antarctica lipase A); iCALB = Novozyme 435 (Candida antarctica lipase B immobilized on acrylic resin); CALB = Roche Chirazyme L-2 (lyophilized Candida antarctica lipase B); CRL = Candida rugosa lipase; EHAD1 = epoxide hydrolase AD1 from Agrobacterium radiobacter; PCL = Amano PS (lyophilized Burkholderia (formerly Pseudomonas) cepacia lipase); PFE = Psuedomonas fluorescens esterase; PLE = pig liver esterase; ROE = Rhizopus oryzae esterase; SABP2 = salicylic acid binding protein 2 from tobacco, an esterase.
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